Medicines for Malaria from the Natural Products

R. Thirumurugan

Anopheles mosquito

Malaria

• Caused by protozoan parasites belonging to the genus Plasmodium

• 4 species – human infections a) Plasmodium falciparum b) Plasmodium vivax c) Plasmodium malariae d) Plasmodium ovale

P. falciparum – severe disease & mortality

P. vivax & P. ovale – form resting stages in the liver (hypnozoites) that, once reactivated, can cause a clinical relapse many months after the initial event

Plasmodium - infect and destroy RBC, leading to fever, severe anaemia, cerebral malaria and, if untreated, death.

Life cycle of malarial parasite

1) Liver stage infection

2) Erythrocyte stage infection

Nature Biotech 2002 & NRDD 2009 8 879

Overview of Antimalarial drugs

NRDD 2009 8 879

N

N

OH

O

N

NH

Cl

N

N

NH

Cl

OH

N

N

NHN

O

N

NHNH2

O

N

NH

OH

CF3

CF3

N

OHCF3

Cl Cl

Cl Cl

Cl

NOH

Quinoline and related antimalarials:

Quinine Chloroquine Amodiaquine

PamaquinePrimaquineMefloquine

Halofantrine Lumifantrine

O

O

O O

O

O

O

O O

OH

O

O

O O

OR

SNH

NN

O

O

NH2

OO

N

NNH2

NH2

Cl

Cl

O

O

OH

Artemisinin DHA

R = Me (Artemether)R = Et (Arteether)R = CO(CH2)2CO2H

(Artesunate)

PyrimethamineSulphadoxine Atovaquine

Overview of Antimalarial drugs

NRDD 2009 8 879

Antimalarial History

Quinine 1820

Quinacrine/ Mepacrine 1935

Chloroquine 1945

Amodiaquine 1947

Proguanil 1948

Pyrimethamine 1951

Pyrimethamine – Sulfadoxine 1965 – 1972

Mefloquine 1984

Halofantrine 1988

Artemether 1994

Atovaquone - Proguanil 1996

Artemether - Lumefantrine 1999, 2009

Chlorproguanil - Dapsone 2003

Artesunate - Amodiaquine 2007

Artesunate - Mefloquine 2008

Global status of resistance

Nature 2002 415 686

1) Without a replacement drug having the low cost & reliability of Chloroquine, morbidity & mortality resurged, notably among children in Africa

2) Resistant P. vivax is present in several regions of southeast Asia & also occur in south America

3) Artemisinin resistance is confirmed in Thailand and Cambodia

Medicines for Malaria Venture (MMV)

• MMV – established in 1999 in Geneva (first public-private partnership)

• Aims to develop one new Antimalarial drug every five years

• MMV solicits, selects and manages discovery and development research at

different institutions. Projects are selected with the help of Expert Scientific

Advisory Committee on a competitive basis and reviewed regularly.

• The product profiles for MMV for uncomplicated malaria include the

following:

1) efficacy against drug-resistant strains

2) cure within 3 days

3) low propensity to generate rapid resistance

4) safe in small children

5) safe in pregnancy

6) appropriate formulations and packaging & low cost of goods.

NRDD 2004 3 509

The collaborations of MMV

Trends Parasitology 2006 22 301

MMV Project Portfolio

Research Translational DevelopmentLead generation

Lead Optimization

Preclinical Phase I Phase IIa Phase IIb / III Registration Phase IV

Novartis MP Novartis series

NITD 609Novartis/MMV

GSK 932121GSK/MMV

ArtemisoneUHKST/MMV

AZCQPfizer/MMV

EurartesimSigma-Tau/MMV

Coartem DispersibleNovartis/MMV

GSK MP PyridoneGSK/MMV

MK 4815MMV/GSK

TafenoquineGSK/MMV

OZ 439Monash/MMV

PyramaxUniv Iowa/MMV

ASAQ Winthrop

Broad & Genzyme MP

DHODHGenzy/MMV

CEM 101 IV ArtesunateGuilin/MMV

Pfizer screening

Genz Aminoindole

P218DHFRBiotec/MMV

Sanofi-aventis Quinoline Methanols

AstraZeneca screening

Genz DHODH

Kinases Oxaboroles

Natural Products

SSJ-183

Antimalarial St.Jude/Rutgers

Aminopyridine

11 other Projects

Pyrazoles

Quinolones

http://www.mmv.org/

Molecules launched & under development

Coartem

Artemether +Lumefantrine

ASAQ

Artesunate +Amodiaquine

Eurartesim

Dihydroartemisinin +Piperaquine

Pyramax

Pyronaridine +Artesunate

AZCQ

Azithromycin +Chloroquine

N O

O

CF3

NHNH2

O

O

H

HO

O

N

SO O

N

N

NH

N

N

OH

O

O

N

NN

N

O

OO

NH

NH2

O NH

OO

OCF3

Br

NH

NH

NH

O

ClF

Cl

Tafenoquine Artemisone Pyronaridine

SSJ 183OZ 277 (OZ 439) GSK 932121 derivative

NITD 609

NITD 609

Preclinical

Project Leader: Dr Thierry Diagana, Novartis Institute for Tropical Diseases, Singapore

MMV Project Director: Dr Julie Lotharius

1. NITD 609 is a novel, synthetic antimalarial molecule - Spiroindolone class, awarded MMV Project of the Year 2009. It is structurally related to GNF 493, a compound first identified as a potent inhibitor of P. falciparum growth in a high throughput phenotypic screen of natural products conducted at the Genomics Institute of the Novartis Research Foundation (GNF) in San Diego, California in 2006.

2. NITD 609 is one of only a handful of molecules capable of completely curing mice infected with P. berghei – a model of blood-stage malaria. Given its good physicochemical properties, promising pharmacokinetic and efficacy profile, the molecule was recently approved as a preclinical candidate and is now entering GLP toxicology studies with the aim of entering Phase I studies in humans in late 2010.

3. If its safety and tolerability are acceptable, NITD 609 would be the first antimalarial not belonging to either the artemisinin or peroxide class to go into a proof-of-concept study in malaria.

http://www.mmv.org/

Spiroazepineindole (GNF 493)

NH

NH

NH

O

Br

Spiroazepineindole

1 (racemate)

NF54 IC50 = 90 nMK1 IC50 = 80 nM

NH

O

NH

NH2

NH

NH

NH

O

BrNaBH3CN, NH4OAc

rt

5-bromoisatin

p-TsOH.H2O

110 oCSpiroazepineindole

1 (racemate)

JMC 2010 53 5155

NH

NH

NH

O

Br

NH

NH

NH

O

Br

1a (1R,3S)

NF54 IC50 = 20 nMK1 IC50 = 30 nM

1b (1S,3R)

NF54 IC50 = > 5000 nMK1 IC50 = > 5000 nM

In-vitro antimalarial activity of SAR of the Spiroindolones

JMC 2010 53 5155

Spiroindolone derivatives

NH

NH

NH

O

Cl

Spiroindolone (racemic)

NF54 IC50 = 27 nMK1 IC50 = 21 nM

NH

NH

NH

O

Cl

NH

NH

NH

O

Cl

NH

NH

NH

O

Cl

NH

NH

NH

O

Cl

(1R,3S)

NF54 IC50 = 9 nMK1 IC50 = 9 nM

(1S,3R)

NF54 IC50 = >5000 nMK1 IC50 = >5000 nM

(1S,3S) (1R,3R)

NF54 IC50 = 1808 nM NF54 IC50 = 444 nM

NH

R1

R2 NH

R1

R2

HO

NH

R1

R2

NO2

NH

R1

R2

NH2

NH

NH

NH

O

ClPOCl3

DMF

Nitroethane

NH4OAc

reflux

LiAlH4

THF

reflux

5-chloroisatin

p-TsOH.H2O

110 oCR1 = F; R2 = HR1 = H; R2 = ClR1 = F; R2 = ClR1 = R2 = F

Spiroindolone (racemic)

JMC 2010 53 5155

Comparison of the pharmacokinetic properties

JMC 2010 53 5155

In vivo efficacy data of NITD 609

Science 2010 329 1175

NH

NH

NH

O

ClF

Cl

Liriodendron tulipifera

• Commonly known as tulip tree or yellow popular

• Family: Magnoliaceae• Native to eastern United States

• Main constituents: Sesquiterpene lactones; Aporphine type alkaloids; Phenyl propanoids• Uses: cytotoxicity & substitute for quinine

Extraction procedure

Dried bark powder (Liriodendron tulipifera) (100gms)

+ 1000 ml Hexane Shake at rt, 24hrs & Filtered

Hexane extract Marc (Air dried)

+ 1000 ml 95% ethanol X 2 Shake at rt, 24hrs & Filtered

Ethanolic extract Marc

CHCl3 Extract Aqueous extract

Discarded

Basify by 5N NH4OH (30ml)pH 8-10 & extracted with CHCl3

Extraction Procedure for the plant Liriodendron tulipifera:

Crude hexane ext

Conc todryness

Acidified CHCl3 Extract

(1.733gm)

CHCl3 extract Aqueous Extract

(85mg)

(IC50 = 6.55 ug/ml)

Conc & dissolved in 3% HCl (200 ml)& extracted with CHCl3 (3X200ml)

Conc todryness

Isolated compounds from the bark of L. tulipifera L

NH

OH

OH

NH

H

OH

O

O

NH

O

OH

O

O

N

O

O

O

NH

H

O

ON

O

O

O

O

O

Asimilobine (1) Norushinsunine (2) Norglaucine (3)

Liriodenine (4) Anonaine (5)Oxoglaucine (6)

Compounds D10: IC50 (µg/mL) Dd2: IC50 (µg/mL) CHO: IC50 (µg/mL) SI RI

1 1.22 ± 0.12 5.76 ± 1.27 > 100 > 82.6 4.8

2 29.55 ± 3.90 30.86 ± 2.32 > 100 > 3.4 1.0

3 21.98 ± 2.84 32.22 ± 1.29 > 100 > 4.6 1.5

4 4.09 ± 0.98 7.86 ± 1.10 8.14 ± 0.01 2.0 1.9

5 1.22 ± 0.04 5.18 ± 0.25 >100 > 82.6 4.2

6 9.07 ± 0.09 20.78 ± 2.92 > 100 > 11.0 2.3

Chloroquine 11.00 ± 2.04 ng/mL 73.79 ± 4.75 ng/mL - - 7.9

Emetine - 0.18 ± 0.02 -

Selectivity index (SI) = IC50 CHO / IC50 D10

Resistance index (RI) = IC50 Dd2 / IC50 D10

Antimalarial activity and cytotoxicity of the compounds from L. tulipifera L

SAR

NH

O

O NH

O

O

OH

N

O

O

O

Anonaine Norushinsunine Liriodenine

Antimarial activity = 1.22 ug/mL

Cytotoxicity = > 100 ug/mL

Antimarial activity = 29.55 ug/mL

Cytotoxicity = > 100 ug/mL

Antimarial activity = 4.09 ug/mL

Cytotoxicity = 8.14 ug/mL

NH

O

O

O

O

N

O

O

O

O

O

Norglaucine Oxoglaucine

Antimarial activity = 21.98 ug/mL

Cytotoxicity = > 100 ug/mL

Antimarial activity = 9.07 ug/mL

Cytotoxicity = NT